WO2001077351A1 - Vecteurs et procedes d'expression de proteine double dans pichia pastoris et escherichia coli - Google Patents

Vecteurs et procedes d'expression de proteine double dans pichia pastoris et escherichia coli Download PDF

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WO2001077351A1
WO2001077351A1 PCT/EP2001/003995 EP0103995W WO0177351A1 WO 2001077351 A1 WO2001077351 A1 WO 2001077351A1 EP 0103995 W EP0103995 W EP 0103995W WO 0177351 A1 WO0177351 A1 WO 0177351A1
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collection
promoter
tag
shuttle vector
expression
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PCT/EP2001/003995
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Angelika LÜKING
Caterina Holz
Hans Lehrach
Dolores Cahill
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces

Definitions

  • the present invention relates to a shuttle vector for expression of nucleic acid in Pichia pastoris and Escherichia coli comprising: a promoter which is a yeast alcohol oxidase (AOX) promoter, a CMV promoter, a tetracycline promoter, or a CMV promoter, an E. coli T7 promoter, a Pichia pastoris autonomously replicating sequence (PARS), and a multiple cloning site.
  • AOX yeast alcohol oxidase
  • CMV promoter a tetracycline promoter
  • PARS Pichia pastoris autonomously replicating sequence
  • the present invention also relates to a host cell transformed/transfected with the shuttle vector of the present invention, to a collection of host cells comprising a collection of shuttle vectors of the invention, and to a method of producing a (poly)peptide comprising culturing the host cell of the present invention under suitable conditions and isolating said (poly)peptide from the culture.
  • the present invention relates to a method of rearraying clones comprising picking the collection of host cells of the present invention; and transferring said collection of host cells in arrayed form to a liquid medium or a solid support, to an array of clones that is obtainable by the method of the present invention, and to a kit comprising the vector, the collection of shuttle vectors, the host cell, the collection of host cells, and/or the array of the present invention, in one or more containers.
  • the yeast Pichia pastoris and the bacterium E. coli are commonly used host systems for the expression of recombinant, heterologous proteins.
  • the choice of the expression host determines the quality of the recombinant protein and is critical.
  • Early successes in the production of heterologous proteins were achieved using the well-studied bacterium E. coll (Itakura, 1977).
  • This prokaryotic expression system is simple to handle and reveals a cost-effective and high-level production of heterologous proteins.
  • expression of some genes often leads to the production of aggregated and denatured proteins, localized in inclusion bodies, with only a small fraction maturing into the desired native form (Marston, 1986; Makrides, 1996).
  • the methylotrophic yeast Pichia pastoris has been developed over the last few years into a powerful expression system for a number of foreign genes (reviewed in Cregg, 1993; Faber, 1995). Its ability for rapid growth at high cell density combined with the strong AOX promoter, has in some cases yielded up to several grams of the heterologous express protein per liter of culture (Cregg, 1993). This eukaryotic system has been used successfully for expression of soluble proteins (Cregg et al., 1993; Romanos, 1995).
  • the technical problem underlying the present invention was to provide methods and means that allow fast and efficient protein expression in both prokaryotic and eukaryotic systems at low costs.
  • the present invention relates to a shuttle vector for expression of nucleic acid in Pichia pastoris and Escherichia coli comprising a promoter which is a yeast alcohol oxidase (AOX) promoter, a yeast CUS1 promoter, a tetracycline promoter, or a CMV promoter, an E. coli T7 promoter, a Pichia pastoris autonomously replicating sequence (PARS), and a multiple cloning site.
  • AOX yeast alcohol oxidase
  • the term "Pichia pastoris autonomously replicating sequence” is described in Cregg, 1985 and allows the vector to be isolated from the cells, without having to be linearised. This increases the transformation efficiency from 1x10 3 per ⁇ g DNA to 1x10 5 per ⁇ g DNA.
  • the vector of the present invention advantageously combines eukaryotic and prokaryotic promoter elements. Furthermore, by integration of a Pichia specific autonomous replicating sequence (PARS), vector linearization is no longer required. Thus small amounts of DNA are sufficient for transformation. Also, high transformation efficiencies up to 10 5 clones per ⁇ g DNA (Cregg, 1985) can be obtained. This is especially useful for the transformation of for instance libraries or high throughput cloning/transformation. Moreover, the PARS sequence enables simple recovery of plasmids from yeast. Additional features include a Terminator Region (AOX-Ter) which is a region in the vector behind the inserted DNA.
  • AOX-Ter Terminator Region
  • the vector of the invention allows proteins to be expressed in high-throughput systems, without the necessity for large-scale sub-cloning.
  • soluble expressed proteins like growth factors or receptors can act as biotherapeutics and may be tested in clinical trials (Wittrup, 1999; Cregg, 1993; Romanos, 1992). It is also envisaged that such proteins will be a better target for the screening of antibodies that can then be used in diagnostic assays by recognizing structural epitopes.
  • the soluble proteins are better sources for crystallization and NMR studies since they show probably a functional folding (Gaasterland, 1998).
  • soluble expressed proteins are more suitable for the generation of native protein chips containing high density arrayed proteins.
  • the dual expression vector of the invention allows for the combination of all advantages of each system, and each system is immediately accessible without the need of time consuming sub-cloning procedures.
  • E. coli produces large amounts of heterologous protein but it is well known to show certain problems like the accumulation and aggregation of proteins in inclusion bodies as a result of differences in the prokaryotic and eukaryotic codon usage.
  • the use of the yeast expression system has the advantage of expression of soluble proteins.
  • proteins can be used to make monoclonal and polyclonal antibodies.
  • the use of denatured purified proteins results in antibodies that may recognize only linear epitopes or motifs.
  • the deficiencies in the bacterial host cell's ability to express soluble proteins and to perform post-translational modifications such as disulfide isomerization, phosphorylation or glycosylation diminish the applicability of bacterial protein expression as an expression system for proteins to be used for the generation and binding characterisation of antibodies or fragments thereof, or other protein binders. Since antibodies are widely used in diagnostic assays, the production of antibodies, and specifically monoclonal antibodies with high affinity and specificity to their antigen is desired.
  • Soluble expressed proteins are also a better source for crystallization and NMR structure analysis since non-water soluble proteins remained an unsolved problem (Gaasterland, 1998).
  • robot technology it is possible to make high density protein arrays on filters (Buessow et al., 1998) or chips (Lueking et al., 1999).
  • Proteins expressed in E. coli can be purified in high-throughput under denatured conditions and can be spotted onto a protein binding surface (Lueking et al., 1999). In high-throughput, soluble proteins expressed in P.
  • pastoris can be arrayed following purification under native conditions to generate a native protein chip, thereby facilitating chip-based binding and interaction (e.g. protein-protein interaction) assays.
  • chip-based binding and interaction e.g. protein-protein interaction
  • MALDI-TOF- MS identification of binding ligands, drugs or compounds by mass spectrometry is envisaged (James et al., 1993; Pappin, 1993; Cottrell, 1994).
  • the vector of the invention is used for the in vitro transcription/translation of cloned nucleic acid molecules, e.g., by using S30 lysate. Such methods are well known in the art (see, e.g., Sambrook et al.).
  • a termination signal such as, e.g., the E. coli T7 termination region may additionally be cloned downstream of the multiple cloning site into the vector of the invention to ensure efficient termination.
  • This embodiment may thus be useful for the production of proteins that are toxic for the host cells used in accordance with the invention.
  • the yeast CUS1 promoter is preferably from S. cerevisiae Koller et al., 2000.
  • the CUS1 promoter from S. cerevisiae leads to a significant reduction of the time of incubation when producing proteins in P. pastoris without, however, reducing the amount of protein produced as compared to the AOX promoter.
  • human GAPDH protein has been expressed using the CUP promoter 100 ng/ml of GAPDH protein was obtained (Koller et al., 2000).
  • the shuttle vector of the invention further comprises a nucleic acid sequence encoding a tag.
  • tag denotes, in accordance with the invention, a proteinaceous or peptidic sequence that can be detected either due to its capacity to emit a signal under suitable and generally established conditions or due to its capacity to react with a detectable further compound such as an antibody specifically recognizing the tag.
  • said tag is an oligo-histidine domain (His 6) ), GST-tag or a hybrid tag comprising his-tag and biotin tag.
  • tags like, e.g., c-myc, FLAG, alkaline phosphatase, EpiTagTM, V5 tag, T7 tag, XpressTM tag, hemaglutinin (HA)-tag, Strep-tag, or biotin, a fusion protein, preferably GST, cellulose binding domain, green fluorescent protein, yellow fluorescent protein, maltose binding protein or lacZ, can also be used in accordance with the invention.
  • a further preferred tag is a hybrid tag of two tags, which are well-known in the art, namely a histidine tag and an in vivo biotinylation recognition site, which functions in both bacteria and yeast (Schatz, 1993; Smith et al., 1997).
  • any of the above tag sequences and in particular the hybrid tag sequence may be followed by a trypsin recognition site which allows a unambiguous identification of proteolytic degradation products in mass spectrometric analysis.
  • a further preferred tag is GST-tag, which allows a very efficient purification from proteins produced in P. pastoris as shown in the figures and the appended examples.
  • said tag is a part of a fusion protein upon expression of said nucleic acid.
  • the tag may advantageously be used for the isolation and/or detection of the produced fusion protein.
  • said tag is positioned N-terminally of the fusion protein.
  • said E. coli T7 promoter is placed downstream from said yeast AOX promoter.
  • the shuttle vector comprises a recombinant insert.
  • said insert is part of a library.
  • said library is a cDNA library.
  • the above-described shuttle vector of the invention comprises at least one, such as two, three, four or five selectable markers.
  • Selectable marker genes useful for the selection of transformed E. coli cells or transformed P. pastoris cells are well known to those skilled in the art and comprise, for example npt, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995), hygro, which confers resistance to hygromycin (Marsh, Gene 32 (1984), 481-485) and amp r which confers resistance to ⁇ -lactam antibiotics like ampicillin (Sykes and Mathew, J. Antimicrob. Chemother.
  • bsd gene derived from Aspergillus terreus which encodes a blasticidin S deaminase and confers resistance to Blasticidin (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338) and the Sh ble gene (Streptoallteichus hindustanus bleomycin gene) which confers resistance to ZeocinTM (Calmes et al., Curr. Genet. 20, 309-314, 1991).
  • the bsd gene as well as the Sh ble gene and the corresponding antibiotic selection agents are commercially available e.g. from Invitrogen Corporation, USA.
  • the shuttle vector of the invention contains a selection marker functioning both in E. coli and P. pastoris.
  • said selection marker is the bsd gene or the Sh ble gene.
  • the present invention relates to a collection of shuttle vectors of the present invention.
  • selection of shuttle vectors denotes a plurality of shuttle vectors of the invention that comprise the same, the same and different, or different recombinant inserts.
  • a collection of shuttle vectors may, e.g., be obtained by preparing a cDNA library and cloning the synthesized cDNAs or cDNA fragments into the shuttle vector of the invention.
  • the present invention also relates to a host cell transformed/transfected with the shuttle vector of the present invention.
  • Methods for introducing nucleic acid molecules into cells are well known in the art and vary depending on the type of host used.
  • calcium chloride transfection is commonly utilized for prokaryotic cells whereas, e.g., calcium phosphate, liposome or DEAE-Dextran mediated transfection or electroporation may be used for eukaryotic host cells (see, e.g., Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y., and Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989)).
  • Electroporation is another method well-known in the art, for transfection in prokaryotic cells (Sambrook et al., 1989).
  • the present invention relates to a collection of host cells comprising the collection of shuttle vectors of the present invention.
  • the term "collection of host cells” refers to at least two host cells, preferably a variety and most preferably a library of host cells that comprise the shuttle vector of the invention with the same, the same and different, or different recombinant inserts.
  • the host cell or the collection of host cells is/are Pichia pastoris cell(s).
  • the host cell or the collection of host cells is/are E. coli cell(s).
  • the present invention relates to a method of producing a (poly)peptide comprising culturing the host cell of the present invention under suitable conditions and isolating said (poly)peptide from the culture.
  • the synthesized fusion protein will be secreted into the culture medium or will accumulate in the cells.
  • the term "culture" as used in accordance with the present invention comprises the culture medium and/or the cells used to synthesize the (poly)peptide of the invention.
  • Suitable culture media culture conditions like, e.g., temperature, and protocols for the recovery of (poly)peptides from the culture medium or from cells, including the disruption or lysis of the cells, are well known to the person skilled in the art (see, e.g., Sambrook et al., loc. cit. and Ausubel et al., loc. cit.).
  • sequences encoding a leader peptide e.g, an alpha leader (for secretion in yeast) and/or, e.g., an ompA leader (for secretion in prokaryotes) may be cloned into the vector of the invention downstream of the T7 promoter.
  • a sequence encoding a protease recognition sequence e.g. for factor X or thrombin may be cloned into the vector downstream of the leader sequences. This may be advantageously used for cleaving off the leader sequences after secretion of the (poly)peptide.
  • the present invention relates to a method of rearraying clones comprising picking the collection of host cells of the present invention; and transferring said collection of host cells in arrayed form to a liquid medium or a solid support.
  • array form refers to any regular or non-regular form that can be replicated. Preferred are regular forms.
  • said arrayed form is a grid form.
  • the grid should preferably allow for the high density array of clones.
  • Suitable grids envisaged in accordance with the present invention are, e.g., a microtitre plate such as a 24 well, a 96 well or a 384 well microtitre plate (standard format: 13cmx8.5cm), a silica wafer, a chip (for example in the standard microscope slide format of 7.5cmx2.5cm), a mass spectrometry target or a matrix as described, e.g., in WO 99/57311 or WO 99/57312.
  • said picking and/or transferring is assisted or effected by automation.
  • Such automated devices may comprise a picking robot and/or spotting robot and/or gridding robot as reviewed in Cahill, 2000.
  • the method further comprises the expression of the recombinant inserts comprised in the shuttle vector contained in the collection of host cells.
  • the present invention relates to an array of clones that is obtainable by the method of the present invention.
  • the present invention relates to a kit comprising the vector; the collection of shuttle vectors; the host cell; the collection of host cells, and/or the array of the present invention, in one or more containers.
  • Suitable containers may be, e.g., vials and the components may, optionally, be. contained in buffers and/or solutions. Additionally or alternatively, one or more of said components may be adsorbed to a solid support such as, e.g., a nitrocellulose filter or nylon membrane, or to the well of a microtitre-plate, a glass slide, a mass spectrometry matrix, a BIAcore chip, a SPR chip, a gel, a coated surface, a gel coated surface, a porous surface, a non-porous surface, a teflon coated surface, a gold coated surface, or a mixture of surfaces.
  • a solid support such as, e.g., a nitrocellulose filter or nylon membrane, or to the well of a microtitre-plate, a glass slide, a mass spectrometry matrix, a BIAcore chip, a SPR chip, a gel, a coated surface, a gel coated surface, a
  • Figure 1 The dual expression vector pZPARS-T7-RGSHis 6 HA, that allows the dual expression in P. pastoris and E. coli.
  • Figure 2 Comparison of protein expression in P. pastoris and E. coli using the dual expression vector.
  • Proteins were expressed in P. pastoris and E. coli and purified under native and denatured conditions. The purified proteins were separated on SDS-PAGE on 12% polyacrylamide gels, electroblotted and detected by immunohistochemie as described in the examples. Lane A: native purified proteins expressed in P. pastoris; lane B: native purified protein expressed by E. coli; lane C: denatured purified proteins expressed by E. coli.
  • Figure 3 Expression in P. pastoris on a 24-well microtitre plate scale. For high-throughput scale expression ten clones were expressed in 24-well microtitre plate and purified as described in the examples. The eluates were separated by SDS-PAGE on a 12% polyacrylamide gel, electroblotted and detected by immunohistochemistry.
  • Lane 1 GAPDH (29); lane 2: HSP90 (12); lane 3: EEF1A1 (9); lane 4: unknown (3); lane 5: human ribosomal protein (25); lane 6: FEZ1 (14); lane 7: unknown (6); lane 8: EEF1A1 (20); lane 9: unknown (clone 26); lane 10: human p47 (8) (abbreviations as generally used as Genbank, public DNA and protein database entry names).
  • FIG. 4 GAPDH expression purified by GST-Sepharose-Beads A gel comparing expression of human GAPDH in pZPARST732-Cup-NST-BT having been induced with 100mM CuSo 4 , and comparing expression in yeast strains GS115 and SMD1168 (a protease deficient strain). The results show that improved protein expression is obtained in the GS115 strain. Also various amounts of GST-Sepharose Beads were tested (as they are expensive) and from this experiment, the optimal amount is 100//I beads, when purifying from 1ml of cell lysate obtained from a 200ml culture volume.
  • Example 1 Strains, transformation and media
  • Escherichia coli strains XL-1 Blue and SCSI (Stratagene) were used for cloning and propagation of the recombinant plasmids and E. coli strain BL21(D3)pLysS (Invitrogen) was used as host for bacterial protein expression.
  • Recombinant plasmids were transformed either by electroporation using a Gene Pulser (Bio-Rad) or by using rubidium chloride competent cells.
  • E. coli transformants were selected on LB medium (0,5% yeast extract, 1% NaCI, 1% bactotryptone) containing 2% dextrose supplemented with 25 /g/ml zeozin.
  • the medium contains additionally 34 ⁇ g/ml chloramphenicol.
  • LB medium supplemented with 1 mM Isopropyl-b-D-thiogalactopyranosid (IPTG) was used.
  • IPTG Isopropyl-b-D-thiogalactopyranosid
  • the Pichia pastoris strain GS115 (his4, Mut+) (Invitrogen) was used for eukaryotic protein expression.
  • the transformation of Pichia pastoris was performed by electroporation (Gene Pulser; Bio-Rad) according to the manufacturer's protocol but, using a resistance of 400 ⁇ instead of 200 ⁇ .
  • Transformants were selected on YPD agar plates (1% yeast extract, 2% peptone, 2% dextrose, 2% agar) supplemented with 100 ⁇ g/ml zeozin. Growth of Pichia pastoris cultures were performed in YPD medium supplemented with 100 or 120 ⁇ g/ml zeozin. For protein expression, Pichia pastoris cultures were transferred to BMMY medium (1% yeast extract, 2% peptone, 1.34% YNB without amino acids, 0.5% methanol) supplemented with 100 ⁇ g/ml zeozin for induction by methanol.
  • BMMY medium 1% yeast extract, 2% peptone, 1.34% YNB without amino acids, 0.5% methanol
  • the T7 expression cassette was transferred as a Avalll/Notl fragment into a modified pPICZa (Invitrogen) vector that was previously deleted for c- myc and 6x (His) tag sequences resulting in pZT7-RGSHisHA.
  • the PARS1 sequence was amplified with the primers PARS1-5 (CTTGGATCCG ATAAGCTGGG GGAACATT) and PARS1-3 (TCCGGATCCA ATTAATATTT ACTTATTTTG GT) from the vector pYM8.
  • the human fetal brain cDNA library hExl
  • hExl human fetal brain cDNA library
  • 2xYT-agar plates 230 mm x 230 mm; Genetics
  • the resulting colonies were scraped from the agar plates and the plasmid DNA was isolated. 10 ⁇ g of hExl plasmid DNA was restricted using Sall/Notl.
  • the DNA was electrophoretically separated and inserts in the range of 0.5 to 3 kb were isolated from the gel, purified (QIAGEN; QIAquick Gelextraction Kit), cloned into the Sall/Notl restricted pZPARS-T7RGSHisHA, transformed into E. coli (strain SCSI) plated out on 2xYT agar plates (230 mm x 230 mm; Genetics) and grown for 20 h at 37 °C. Approximately 100,000 colonies were scraped from the agar and plasmid DNA isolation was performed. This DNA preparation was used for transformation of P. pastoris.
  • each well was filled with 200 ⁇ l YPD supplemented with zeozin (100 ⁇ g/ml) and grown for three days at 30 °C.
  • the inserts were amplified by PCR using the primers AOX5 (TTGCGACTGG TTCCAATTGA CAAG) and AOX3 (CATCTCTCAG GCAAATGGCA TTCTG).
  • PCR amplification cells of each clone was transferred twice using a 96-well Nunc replicator into an 50 ⁇ l reaction mix (50 mM KCI, 35 mM Tris-Base, 15 mM Tris-HCI, 1.5 mM MgCI, 0.1% Tween 20, 0.2 mM dWP ' s, 3 units Taq, 0.1 units lyticase (Sigma L2524)).
  • the PCR was performed as follows: 30 min./37 °C; 4 min./94 °C; for 30 cycles: 45 se ⁇ /94 °C, 20 sec./ 55°C, 2.30 min. ⁇ 72 °C; 10 min./72°C.
  • the PCR products were purified and sequenced. Sequences analysis was performed using MacMolly Tetra (SoftGene GmbH) and by blasting against the public sequence databases (NCBI).
  • Plasmid preparations were done from confirmed XL-1Blue clones (QIAquick Spin Miniprep Kit; QIAGEN) and 10 ⁇ l of DNA was transformed in BL21 (D3)pLysS by using the rubidium chloride method (Lueking et al., 2000).
  • Escherichia coli Proteins were expressed in E. coli (strain BL21 (D3)pLysS) liquid cultures. 2 ml LB medium containing 25 ⁇ g/ml zeozin and 34 ⁇ g/ml chloramphenicol were inoculated with 0.5 ml of an overnight culture and were shaken at 37 e C until an OD 6 oo of 0.8- 1.0 was reached. IPTG was added to a final concentration of 1 mM. The culture was shaken for 5 h at 37 Q C and cooled to 4 Q C on ice.
  • phosphate buffer 50 mM NaH PO 4 , 300 mM NaCI, pH 8.0.
  • the cells were then lysed in 3 ml per gram wet weight of lysis buffer (50 mM Tris, 300 mM NaCI, pH 8.0) containing 10 mM imidazol, 1 mM PMSF, 0.25 mg/ml lysozyme, 1 mg/ml RNAse and 1 mg/ml DNAse at 4° C over night.
  • the lysate was cleared by centrifugation at 10,000 g for 10 min.
  • Ni-NTA agarose (Qiagen) was added and mixed by shaking at 4 S C for 1 h. The mixture was poured into a unpacked column and subsequently washed with ten bed volumes of lysis buffer containing 20 mM imidazole. Protein was eluted in lysis buffer containing 250 mM imidazole.
  • cells were harvested by centrifugation at 2,100 g for 5 min. and were washed in 1 ml phosphate buffer (50 mM NaH 2 P0 , 300 mM NaCI, pH 8.0). The cells were then lysed in 500 ⁇ l buffer B (8 M urea, 100 mM NaH 2 PO4, 10 mM Tris pH 8.0) for 30 min. at room temperature. Bacterial debris were pelleted by centrifugation at 10,000 g for 10 min. Ni-NTA agarose (QIAGEN) were added to the supematants and mixed by shaking at room temperature for 30 min.
  • phosphate buffer 50 mM NaH 2 P0 , 300 mM NaCI, pH 8.0
  • 500 ⁇ l buffer B 8 M urea, 100 mM NaH 2 PO4, 10 mM Tris pH 8.0
  • Ni-NTA agarose was then pelleted by centrifugation at 10,000 g for 5 min., washed with 1 ml buffer C (8 M urea, 100 mM NaH 2 PO 4 , 10 mM Tris pH 6.3) followed by elution of the proteins with 100 ⁇ l buffer E (8 M urea, 100 mM NaH 2 PO4, 10 mM Tris pH 4.5).
  • Pichia pastoris Proteins were expressed in P. pastoris (strain GS115) in liquid cultures. 20-100 ml BMMY medium (as described in Sambrook et al., 1989) was inoculated with 2 ml of a P.
  • clones were cultivated in 24-well microtitre plate with a culture volume of 5 ml. Cells were harvested by centrifugation at 2,100 g for 5 min, washed in 1 ml phosphate buffer (50 mM NaH 2 PO 4 , 300 mM NaCI, pH 8.0) and re-suspended in 0.3 ml lysis buffer (50 mM Tris, 300 mM NaCI, pH 8.0) containing 10 mM Imidazol, 1 mM PMSF.
  • phosphate buffer 50 mM NaH 2 PO 4 , 300 mM NaCI, pH 8.0
  • lysis buffer 50 mM Tris, 300 mM NaCI, pH 8.0
  • the cells were mixed with an equal volume of glass beads (size 0.5 mm), vortexed for 30 seconds and incubated on ice for 30 seconds. This procedure was repeated seven times and the lysate was cleared by centrifugation at 10,000 g for 5 min. Ni-NTA agarose (QIAGEN) was added and mixed by shaking at 4 Q C for 1 h. The mixture was poured onto an unpacked column which was subsequently washed with ten bed volumes of lysis buffer containing 20 mM imidazole. Protein was eluted with lysis buffer containing 250 mM imidazole.
  • the purified proteins were separated by SDS/ PAGE (12.5 %) and transferred onto PVDF membrane (Millipore) by electroblotting (T77 SemiPhor, Pharmacia Biotech). After blotting, the filters were washed in TBST (TBS, 0.1 % (v/v) Tween 20) for 1 min, blocked in 2 % (w/v) bovine serum albumin (BSA)/ TBST for 60 min. and incubated with the monoclonal antibody ⁇ -RGSHis 6 (Qiagen) diluted 1 :2000 in 2 % (w/v) BSA/ TBST for 1 h at room temperature. This was followed by two 10 min.
  • the expression shuttle vector consists of the yeast alcohol oxidase (AOX) promoter, followed by the T7 promoter region including the bacterial ribosome binding site and a translation initiation site enabling transcription and expression in both hosts.
  • the vector contains an amino-terminal oligo-histidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites.
  • PARS1 Pichia specific autonomous replicating sequence
  • vector linearization is no longer required, increasing the transformation efficiency up to 10 5 per ⁇ g DNA (Cregg, 1985).
  • the PARS1 sequence allows the recovery of plasmids from yeast enabling a simple propagation from yeast to bacteria by transformation. Due to the use of a common selection marker zeozin, the size of the shuttle vector remains small resulting in convenient handling, cloning and transformation (Fig. 1).
  • Example 9 Construction and characterization of a human fetal brain (hExl) expression library in the dual expression vector
  • the hExl cDNA expression library (Buessow, 1998), which was re-arrayed based on its expression subset in E. coli, was sub-cloned into this novel dual expression vector and was then transformed into P. pastoris.
  • 96 randomly chosen P. pastoris clones were picked and analyzed by PCR, DNA sequencing and protein expression. An average cDNA insert size of 1.6 kb was determined. 5 ' -tag sequences of 95 clones were obtained and used for BLASTN searches against the public databases including Genbank and Unigene databases (Altschul, 1997). 83 (87 %) clones were found to match human proteins and 12 (13 %) clones were representing unknown sequences.
  • Plasmids from the 29 P. pastoris expression clones were isolated and shuttled by transformation into E. coli. Expression products of all clones from both hosts were affinity-purified under native and denaturing conditions using Ni-NTA agarose. 29 (100 %) expression products were purified from P. pastoris in a soluble form. 25 (86.2 %) E. coli expressed proteins have been purified under denaturing conditions, whereas 8 (27.6%) proteins have been isolated in a soluble form, partly detected by sensitive detection methods, such as western immunoblotting (Fig. 2). Protein concentrations have been determined for each purified expression product and concentration in the range of 1.5-20 ⁇ g/ml or purified protein (denatured) were obtained from E. coli and in the range of 0.1 -5 ⁇ g/ml (native) when expressed in P. pastoris.
  • Table 1 Protein expression properties of the expression clones with the estimated protein weight.

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Abstract

La présente invention concerne un vecteur navette servant à l'expression d'acide nucléique dans Pichia pastoris et Escherichia coli, ledit vecteur comprenant : un promoteur qui est un promoteur de l'alcool oxydase (AOX) de levure, un promoteur CUS1 de levure, (ac) un promoteur tétracycline ; ou (ad) un promoteur CMV, un promoteur E. coli T7 ; une séquence de réplication autonome de Pichia pastoris (PARS) ; et un site de clonage multiple. Cette invention concerne également une cellule hôte transformée/transfectée au moyen du vecteur navette de l'invention, un ensemble de cellules hôtes comprenant un ensemble de vecteurs navettes de l'invention, et un procédé permettant de produire un (poly)peptide, ledit procédé comprenant la culture de la cellule hôte de l'invention dans des conditions appropriées, et l'isolation dudit (poly)peptide de la culture. Pour finir, l'invention a pour objet : un procédé permettant de redisposer les clones en jeu ordonné, ledit procédé comprenant le prélèvement de l'ensemble de cellules hôtes de l'invention, et le transfert dudit ensemble de cellules hôtes mis sous forme de jeu ordonné, dans un milieu liquide ou un support solide ; un jeu ordonné de clones pouvant être obtenu grâce au procédé de l'invention ; et un ensemble comprenant le vecteur, l'ensemble de vecteurs navettes, la cellule hôte, l'ensemble de cellules hôtes, et/ou le jeu ordonné de la présente invention, dans un ou plusieurs récipients.
PCT/EP2001/003995 2000-04-07 2001-04-06 Vecteurs et procedes d'expression de proteine double dans pichia pastoris et escherichia coli WO2001077351A1 (fr)

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US7741098B2 (en) * 2001-11-27 2010-06-22 Nexyte Ab Production of eukaryotic proteins and nucleic acid molecules in C. elegans
CN108998466A (zh) * 2018-08-06 2018-12-14 天津农学院 一种酵母中高效克隆载体的构建方法及应用
EP3592800A4 (fr) * 2017-03-10 2021-01-06 Bolt Threads, Inc. Compositions et procédés de production de rendements sécrétés élevés de protéines recombinées
US11370815B2 (en) 2017-03-10 2022-06-28 Bolt Threads, Inc. Compositions and methods for producing high secreted yields of recombinant proteins
US12122810B2 (en) 2022-05-23 2024-10-22 Bolt Threads, Inc. Compositions and methods for producing high secreted yields of recombinant proteins

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7741098B2 (en) * 2001-11-27 2010-06-22 Nexyte Ab Production of eukaryotic proteins and nucleic acid molecules in C. elegans
EP3592800A4 (fr) * 2017-03-10 2021-01-06 Bolt Threads, Inc. Compositions et procédés de production de rendements sécrétés élevés de protéines recombinées
US11306127B2 (en) 2017-03-10 2022-04-19 Bolt Threads, Inc. Compositions and methods for producing high secreted yields of recombinant proteins
US11370815B2 (en) 2017-03-10 2022-06-28 Bolt Threads, Inc. Compositions and methods for producing high secreted yields of recombinant proteins
US11725030B2 (en) 2017-03-10 2023-08-15 Bolt Threads, Inc. Compositions and methods for producing high secreted yields of recombinant proteins
CN108998466A (zh) * 2018-08-06 2018-12-14 天津农学院 一种酵母中高效克隆载体的构建方法及应用
US12122810B2 (en) 2022-05-23 2024-10-22 Bolt Threads, Inc. Compositions and methods for producing high secreted yields of recombinant proteins

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